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Using the illumina mRNA Seq kits I created 8 libraries last week; they are completed through pcr enrichment and ready to run on the GAs. For the most part the traces looked good however on all 8 samples there is an odd peak at around 75 bps. When I performed a gel cut out at ~300 bps I don't believe I introduced any other material so I am not sure where the extra peak is coming from. It seems to me that it could be a primer dimer from pcr enrichment or some sort of contamination in the gel of my bioanalyzer kit, either way I don't think those would affect the reads on a GA but I'd like to hear some input from you guys. The scans are attached bellow
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If you used a TruSeq kit to construct these, then my guess is that the ~75 "bps" peak is actually the PCR primers used for enrichment PCR. These primers, from the "PPC" (PCR primer cocktail) tube give a peak in that region of the electropherogram of both DNA high sensitivity chips and RNA (pico) chips. See:
We don't run the DNA1000 chips in our lab, so have never looked at it on that type of chip.
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Phillip
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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